The present invention relates to fungal histidine kinases. In particular, the present invention is directed to histidine kinases from Neurospora (e.g., N. crassa), Candida (e.g, C. albicans), and Aspergillus (e.g., A. fumigatus), and related compositions. Furthermore, the present invention provides compositions and methods for the identification of compounds having antifungal activity, as well as compositions and methods for the treatment of fungal infections.
Fungal organisms have become increasingly significant pathogens in immunocompromised patients, especially those who because of cancer, organ transplantation, chemotherapy, pregnancy, age, diabetes, complications following extensive surgery, and various immune system dysfunctions, are at risk of experiencing life-threatening diseases caused by organisms which do not ordinarily pose a threat to normal, immunocompetent people. Indeed, immunocompromised patients perhaps provide the greatest challenge to modern health care delivery. For example, fungal infections have become one of the leading factors contributing to morbidity and mortality in cancer patients, and fungi account for 4-12% of nosocomial (hospital-acquired) pathogens in leukemia patients (E. Anaissie, Clin. Infect. Dis., 14[Suppl.1]:S43 [1992]). The incidence of nosocomial bloodstream infections with fungi such as Candida (xe2x80x9ccandidemiaxe2x80x9d) has increased in recent years and now accounts for 5.6% of all primary bloodstream infections (Id.). There are an estimated 200,000 patients/year who acquire nosocomial fungal infections, with bloodstream infections having a mean mortality rate of 55% (See e.g., Beck-Sague et al., J. Infect. Dis., 167:1247 [1993]; and the Centers for Disease Control website at www.cdc.gov/ncidod/publications/brochures/hip.html). Fungi infections may also be life-threatening in other settings, such as in the case of intravenous drug abusers who use non-sterile substances to dilute drugs prior to injection.
Deep-seated mycoses are being increasingly observed in patients undergoing organ transplants and aggressive chemotherapy. For solid organ transplantation, the incidence of fungal infections ranges from 5% in kidney recipients, 15-25% in lung and heart recipients, and up to 40% in liver recipients (Alexander et al., Drugs 54:657 [1997]). The most common fungal pathogens are the opportunistic yeast, C. albicans and the filamentous mold, A. fumigatus (See, Bow, Br. J. Haematol., 101:1 [1998]; and Warnock, J. Antimicrob. Chemother., 41:95 [1998]).
AIDS patients are also at great risk for fungal infections. An estimated 80% of AIDS patients acquire fungal infections and suffer a mortality rate of 10-20% as a result of these infections (See, Cairns, J. Electron Microsc. Techn. 8:115 [1988]).
Candida
Of the over 100 Candida species, approximately seven are isolated with great frequency from human specimens (T. Mitchell, in Zinsser Microbiology, W. K. Joklik, et al., [eds], Appleton, Century-Crofts, Norwalk, Conn., pp. 1183-1190 [1984]). A brief taxonomic chart of Candida is shown in Table 1. Some Candida species are related to organisms, such as Saccharomyces in the subclass Hemiascomycetidae, class Ascomycetes, subdivision Ascomycotina, division Ascomycota. Also, some mycologists consider C. stellatoidea to be a variant of C. albicans. 
The first exposure to fungi experienced by many humans occurs during the birth process, when C. albicans present in the mother""s vaginal canal colonizes the buccal cavity, and portions of the upper and lower gastrointestinal tract of the newborn. This colonization usually results in the establishment of C. albicans as a commensal organism in these areas, for the life of the individual. However, C. albicans is also the most common fungal pathogen of humans, worldwide, with other Candida species becoming increasingly important in fungal disease in humans and other animals.
As a commensal, C. albicans exists as a unicellular yeast; during invasive disease, the organism has a filamentous morphology. The implication of C. albicans in disease may indicate that the patient has a co-existing immune, endocrine or other debilitating disorder that must also be addressed in order to effectively manage the fungal disease. The principal risk factors that predispose individuals to deeply invasive candidiasis include protracted course of broad spectrum antimicrobials, cytotoxic chemotherapy, corticosteroids, and vascular catheters.
Candida Infection and Diagnosis
Because clinical Candida infections and disease may be acute or chronic, superficial or disseminated, the disease syndromes are many and varied. While C. albicans is most commonly implicated, various other Candida species can and do invade most organ systems of the body. For example, C. tropicalis, C. parapsilosis, C. guilliermondi, C. krusei, and C. lusitaniae have emerged as important pathogens in cancer patients (E. Anaissie, supra). Candidiasis due to C. albicans, as well as other Candida species, is the most common opportunistic fungal infection observed (See, Walsh and Dixon, xe2x80x9cSpectrum of Mycoses,xe2x80x9d in Baron (ed.), Medical Microbiology, 4th ed, University of Texas Medical Branch, Galveston, Tex. [1996], pp. 919-925). Superficial candidiasis may involve the epidermal and mucosal surfaces (e.g., the oral cavity, pharynx, esophagus, intestines, urinary bladder, and vagina). In deep candidiasis, the gastrointestinal tract and intravascular catheters are the two major portals of entry, with the kidneys, liver, spleen, brain, eyes, heart, and other tissues being the major sites involved.
The major difficulties in treating Candida infections are encountered in cases of systemic disease. Chronic mucocutaneous, pulmonary candidiasis, endocarditis, and fungemia must be diagnosed early and treated promptly with an appropriate antifungal regimen, in order to avoid fatality (W. Chandler, Color Atlas and Text of Histopathology of Mycotic Diseases, p. 44 [1980]). The incidence of candidiasis in certain patient populations is striking. Up to 30% of leukemia patients acquire systemic candidiasis (E. Anaissie, supra). This is of great significance, as some reports indicate that the fatality rate for disseminated candidiasis in cancer patients is 80% (F. Meunier, et al., Clin. Infect. Dis., 14[Suppl. 1]:S120 [1992]). Also, fatalities in most organ transplant patients who succumb to infection are most often due to opportunistic organisms, of which Candida is the leading mycotic agent (T. Mitchell, supra).
The probability of postoperative systemic candidiasis is related to the length of operation and may involve contamination with organisms during the surgery or contamination through such diverse postoperative procedures as indwelling catheters or the use of prophylactic antibacterial compounds (Id.). Prosthetic devices, including artificial heart valves or intravenous lines can be colonized and introduce Candida into the patient""s bloodstream (Id.).
Significantly, many patients who develop systemic candidiasis were given corticosteroids prior to development of their Candida infection. Corticosteroids are known to depress the immune system and are often used to prevent transplant rejection. Corticosteroids and antibacterials predispose to candidiasis by depressing phagocytic activity and cell-mediated immunity, reducing the bacterial flora and indirectly increasing the Candida population (Id.). Perhaps also importantly, corticosteroids have been hypothesized to directly act on fungi and may contribute to disease progression in patients with systemic candidiasis (See e.g., D. S. Loose et al., J. Gen. Microbiol., 129:2379 [1983]; D. S. Loose and D. Feldman, J. Biol. Chem., 257:4925 [1982]).
Because of the delays necessary in making a definitive diagnosis, physicians usually treat patients empirically. For superficial infections, topical antifungals are often used; the prognosis for most types of these infections is usually quite good. For systemic disease, highly toxic antifungals must often be used. Administration of these compounds requires careful patient monitoring (patients are usually admitted) because of their serious side effects (e.g., the nephrotoxicity, hypokalemia, anemia, fever and other toxic effects associated with the use of amphotericin B).
Aspergillus
Aspergillus species, as well as various other opportunistic hyaline molds have long been associated with infection and disease in humans and other animals. Indeed, in patients with positive fungal cultures, Aspergillus species are the second most common isolate, after Candida species (See, Kennedy and Sigler, xe2x80x9cAspergillus, Fusarium, and Other Opportunistic Moniliaceous Fungi, in Murray et al., (eds), Manual of Clinical Microbiology, 6th ed., ASM Press, Washington, D.C. [1995], pp. 765-790); and Goodwin et al., J. Med. Vet. Mycol., 30:153 [1992]). There are multiple members of the genus Aspergillus, including the A. fumigatus group, A. niger, A. flavus, and many others (e.g., A. candidus, A. carneus, A. clavatus, A. deflectus, the A. fischeri group, A. flavipes, the A.glaucus group, the A. nidulans group, A. ochraceus, A. oryzae, A. parasiticus, A. restrictus, A. sydowii, A. terreus, A. ustus, and A. versicolor). In addition to these organism names, telomorphic stages of the Aspergillus species are also included in the Eurotium, Emericella, and Neosartorya species.
A. fumigatus is the most frequently observed fungus in airborne spore surveys (Armstrong et al., Issues Mycol., 2:1-20 [1997]). The organism is essentially ubiquitous, as it is capable of growing in a variety of environments, including air ducts, houseplant soil, compost piles, etc. In addition, the organism is capable of growing over a wide temperature range, from xe2x88x9212xc2x0 C. to 50-55xc2x0 C. (See e.g., Conney and Emerson, Thermophilic Fungi: An Account of Their Biology, Activities and Classification, Freeman and Co., San Francisco [1964]). Of the members of the genus Aspergillus, A. fumigatus is the most common human pathogen. Three main types of disease have been associated with A. fumigatus, namely allergic asthma, aspergilloma, and invasive aspergillosis (See e.g., Lortholary et al., Amer. J. Med., 95:177-187 [1993]). Allergic pulmonary asthma due to A. fumigatus exposure affects an estimated 50,000 individuals in the U.S. Most cases are successfully treated with anti-asthma medication and their episodes are self-limiting. Aspergillomas are formed when fungal spores germinate in situ in tissue such as the lungs and form fungus balls. Aspergilloma patients often cough up hyphal plugs. There is no invasion of underlying tissues and in most cases, treatment involves the simple surgical removal of the aspergilloma. Invasive aspergillosis involves the invasion of host tissues, and is most commonly observed in patients with predisposing conditions (e.g., immunosuppressive drugs, neutropenia, chemotherapy, AIDS). Transplant (e.g., bone marrow or organ) and chemotherapy patients are at the greatest risk for this form of aspergillosis (See e.g., Denning et al., New Eng. J. Med., 324:654-662 [1992]; and Miller et al., Chest 105:37-44 [1994]). Aspergillosis is presumptively diagnosed when there is an unexplained pulmonary infiltrate, a patient is unresponsive to antibacterials and/or there is a fever of unknown origin. The prognosis for patients with invasive disease is particularly grave, with mortality rates  greater than 50% (See e.g., Polis et al., xe2x80x9cFungal Infections in Patients with the Acquired Immunodeficiency Syndrome,xe2x80x9d in DeVita et al. (eds), AIDS: Biology, Diagnosis, Treatment, and Prevention, 4th ed., Lippincott-Raven, [1997]), due to the lack of a rapid diagnostic method to confirm A. fumigatus infection, and the lack of safe antifungal drugs.
Fungal Physiology and Treatment of Fungal Diseases
The development of effective antifungal agents has lagged behind that of antibacterial agents. As bacteria are prokaryotic and offer numerous structural and metabolic targets that differ from humans, we have been more successful in identifying and developing antibacterial agents. In contrast, like humans, fungi are eukaryotic.
Thus, most agents toxic to fungi are also toxic to humans. In addition, because fungi generally grow more slowly and in multi-cellular forms in vitro, they are more difficult to quantify than bacteria, complicating experiments designed to evaluate the in vitro and/or in vivo properties of potential antifungals.
Four general groups of antifungals have been developed, including the polyenes (e.g., amphotericin, nystatin, and pimaricin), azoles (e.g., fluconazole, itraconazole, and ketoconazole), allylamines and morpholines (e.g., naftifine and terbinafine), and antimetabolites (e.g., 5-flurocytosine). The site of action for most antifungals is the ergosterol present in the fungal cell membrane, or its biosynthetic pathway. However, other antifungals act at other sites, such as the fungal cell wall.
Fungal Cell Wall
The fungal cell wall is a rigid, stratified structure that consists of chitinous microfibrils encased in a matrix of small polysaccharides, proteins, lipids, inorganic salts, and pigments, that provides support and shape to the cell. The chitin within fungal cell walls is a (xcex21-4)-linked polymer of N-acetyl glucosamine, polymerized by chitin synthase at the plasma membrane.
The major polysaccharides of the cell wall matrix consist of non-cellulosic glucans, including glycogen-like compounds, mannans (mannose polymers), chitosan (glucosamine polymers), and galactans (galactose polymers). Fucose, rhamnose, xylose, and uronic acids may also be present in small amounts. The term xe2x80x9cglucanxe2x80x9d is used in reference to a large group of D-glucose polymers with glycosidic bonds. Of these, the most common glucans present in the fungal cell wall are in the xcex2-configuration. Polymers with (xcex21-3)- and (xcex21-6)-linked glucosyl units with various proportions of 1-3 and 1-6 linkages are common cell wall components. Many fungi, and yeasts in particular, have soluble peptidomannans within a matrix of xcex1- and xcex2-glucans, as part of the outer portion of their cell wall. The fungal cell wall is essential for the viability of the organism, as it prevents osmotic lysis of the cell. Even a small lesion within the cell wall can lead to the extrusion of cytoplasm due to the internal pressure within the cell (See, Cole, xe2x80x9cBasic Biology of Fungi, in Baron (ed.), Medical Microbiology, 4th ed., University of Texas Medical Branch, Galveston, Tex. [1996], pp. 903-911). Indeed, creation of such lesions is the mechanism of action of many antifungal compounds.
As C. albicans may be in the form of budding yeast cells, pseudohyphae, germ tubes, true hyphae, and chlamydospores, differences between these forms are of interest in the development of antifungals. In the yeast form, the C. albicans cell wall contains approximately 30-60% glucan, 25-50% mannan (mannoprotein), 1-2% chitin, 2-14% lipid, and 5-15% protein (McGinnis and Tyring, xe2x80x9cIntroduction to Mycology,xe2x80x9d in Baron (ed.), Medical Microbiology, 4th ed., University of Texas Medical Branch, Galveston, Tex. [1996], pp. 893-902). Glucans with (xcex21-3) and (xcex21-6)-linked groups comprise a high percentage of the yeast cell wall. These glucans may impede the access of amphotericin B to the plasma membrane (See, McGinnis and Tyring, supra, at p. 896).
For filamentous fungi (e.g., Aspergillus) cell wall synthesis and assembly occur only at each hyphal apex, while for yeasts, extension occurs at the bud tip, followed by intercalary growth (Madden et al., Ann. Rev. Microbiol., 52:687 [1998]; and Trinci et al., J. Gen. Microbiol., 103:243-248 [1977]). Chitin and other carbohydrate polymers, with the exception of (1,6)xcex2-glucan, are synthesized de novo at the hyphal tips. Although it is incompletely understood, the assembly of fungal cell walls can be simplistically divided into five steps, including cell wall precursor synthesis, cell wall polymer synthesis, cell wall polymer assembly, morphogenesis, and regulation.
Fungal Cell Membrane
The fungal plasma (i.e., cell) membrane is similar to mammalian plasma membranes, with the exception being that fungal plasma membranes contain ergosterol, rather than cholesterol as the principal sterol. The plasma membrane is selectively permeable, and apparently regulates the passage of materials into and out of the cell. Sterols present in the membrane provide structure, modulate membrane fluidity, and may control other physiologic events.
Polyene antifungals (e.g., amphotericin B, nystatin, and pimaricin) bind ergosterol, to form complexes that allow the rapid leakage of cellular potassium, other ions, and small molecules out of affected fungal cells. This results in the inhibition of fungal glycolysis and respiration. Other antifungals such as the azoles (e.g., fluconazole, imidazole, ketoconazole, and itraconazole), and allylamines and morpholines interfere with the ergosterol biosynthesis. Inhibition of ergosterol formation may result in permeability changes in the plasma membrane, growth inhibition, and may lead to excessive chitin production and abnormal fungal growth.
Fungal Microtubules
Fungi also possess microtubules composed of tubulin. These structures are involved in the movement of organelles, chromosomes, nuclei, and Golgi vesicles. Microtubules are the principal components of the spindle fibers that aid movement of the chromosomes during mitosis and meiosis. Exposure to some antifungal agents disrupts the movement of the nuclei, mitochondria, vacuoles, and apical vesicles. In addition, destruction of cytoplasmic microtubules interferes with transport of secretory materials, and may inhibit cell wall synthesis. Griseofulvin, a compound commonly used to treat dermatophytic infections binds with microtubule-associated proteins involved in tubulin assembly, and acts by stopping mitosis at metaphase.
Antifungal Compounds
Despite the identification of cell membrane, cell wall, and microtubule targets for antifungal action, antifungal development has been slow. Amphotericin B remains the treatment mainstay for life-threatening and other mycoses. Discovered in 1956, amphotericin B remains the drug of choice for candidiasis, cryptococcosis, aspergillosis, zygomycosis, coccidioidomycosis, histoplasmosis, blastomycosis, and paracoccidioidomycosis. Amphotericin B must be administered intravenously and is associated with numerous, often serious side effects, including phlebitis at the infusion site, fever, chills, hypokalemia, anemia, and nephrotoxicity). Importantly, fungal resistance to amphotericin B has been reported for various opportunistic fungi, including Pseudallescheria boydii, Fusarium, Trichosporon, and some C. lusitaniae and C. guilliermondii isolates (See, Dixon and Walsh, xe2x80x9cAntifungal Agents,xe2x80x9d in Baron (ed.), Medical Microbiology, 4th ed., University of Texas Medical Branch, Galveston, Tex. [1996], pp. 926-932).
Nystatin is another broad-spectrum polyene antifungal. However, its toxicity to humans prevents its widespread use. Currently, it is limited to topical applications, where it is effective against yeasts, including C. albicans. Pimaricin (natamycin) is a topical polyene active against yeasts and molds; this compound is used to treat superficial mycotic eye infections.
Ketoconazole was the first antifungal developed that was suitable for oral administration, although it may also be used topically. In non-immunocompromised patients, it may be used to treat histoplasmosis and blastomycosis. It is also used against mucosal candidiasis and various cutaneous mycoses (e.g., dermatophyte infections, pityriasis versicolor, and cutaneous candidiasis). However, it is not useful for treatment of aspergillosis or systemic yeast infections. The triazoles (e.g., fluconazole and itraconazole) have found use in systemic mycoses. Fluconazole is now often used to treat candidemia in non-neutropenic patients, and may be used in cryptococcosis and some cases of coccidioidomycosis. Itraconazole is often effective against histoplasmosis, blastomycosis, sporotrichosis, coccidioidomycosis, and some cases of cryptococcosis and aspergillosis.
Side effects are not as common with the azoles as with amphotericin B, although life-threatening hepatic toxicity may result from long-term use. Other side effects include nausea, vomiting, and drug interactions with such compounds as cyclosporin, antihistamines, anticoagulants, anti-seizure and oral hypoglycemic medications, as well as other compounds, are of potential concern. In addition, the emergence of clinically-resistant strains has raised additional concerns with these compounds (Boschman et al., Antimicrob. Agents Chemother., 42:734 [1998]; and Graybill, Clin. Infect. Dis., 22(Suppl.2):S166 [1996]).
Unlike antibacterials, few antimetabolite compounds are useful as antifungals. The most commonly used antifungal is 5-fluorocytosine, a fluorinated analog of cytosine. However, like other antimetabolites, the emergence of drug resistance has become a problem. Thus, it is seldom used alone. Nonetheless, in combination with amphotericin B, it remains the treatment of choice for cryptococcal meningitis, and is effective against some diseases caused by dematiaceous fungi.
Other antifungals include griseofulvin, an antimicrobial produced by Penicillium griseofulvin, that is active against most dermatophytes. Potassium iodide is another compound that is used as an antifungal to enhance transepidermal elimination of fungal organisms in cases of cutaneous and lymphocutaneous sporotrichosis, although it is not effective against Sporothrix schenckii in vitro.
Antifungal susceptibility testing is generally not standardized, and the results of in vitro tests do not always correspond to the in vivo results. Thus, preliminary antifungal selection is often made on the basis of the specific organism identified as being involved in the patient""s disease. While this approach may be useful in avoiding selection of an antifungal to treat fungi known to exhibit primary resistance to an agent, it is less useful in the selection of antifungals to treat fungi known to develop secondary (i.e., drug-induced) resistance.
Primary, as well as secondary, antifungal resistance has become an increasing problem. For example, with most polyenes, the resistance is almost always primary (i.e., the susceptibility profiles for the species are characteristic, inherent, and rarely change in response to drug exposure). Primary and secondary resistance to azoles has been reported for most medically important yeasts. In view of the development of resistance, as well as the relative lack of variety available in the selection of antifungals, there remains a need for the development of compounds useful for treatment of fungal diseases.
The present invention relates to osmosensing histidine kinases and methods for their use in screening antifungal compounds for their activity against fungal organisms, as well as for the development of antifungal compounds.
In one embodiment, the present invention provides a purified and isolated nucleic acid sequence encoding at least a portion of an osmosensing histidine kinase, wherein the sequence is selected from the group consisting of SEQ ID NO: 1, 3, 4 and 16, and their complementary sequences. In one preferred embodiment, the nucleic acid sequence is from Neurospora. In alternative embodiments, the present invention provides compositions comprising the nucleic acid sequences selected from the group consisting of SEQ ID NO: 1, 3, 4 and 16, and their complementary sequences.
The present invention also provides polynucleotide sequences that hybridize under stringent conditions to the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 3 and 16.
The present invention further provides substantially purified proteins comprising the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 5, 10, 11 and 17.
The present invention also provides expression vectors that contain nucleic acid sequences selected from the group consisting of SEQ ID NOS: 1, 3, 4 and 16, and their complementary sequences. It is contemplated that these sequences may be encoded by one or more expression vectors. It is also contemplated that the expression vectors of the present invention be present within host cells. In one embodiment, the host cell is prokaryotic, while in preferred embodiments, the host cell is eukaryotic. In particularly preferred embodiments, the host cell is a fungal cell. In alternate preferred embodiments the host cell is selected from the group consisting of Neurospora, Candida, Aspergillus, and Saccharomyces. In yet another particularly preferred embodiment, the host cell comprises Candida albicans. 
The present invention also provides host cells comprising at least one DNA sequence selected from the group consisting of SEQ ID NOS: 1, 3, 4 and 16, or a portion thereof, such that the host cell expresses a fungal histidine kinase or a portion thereof. In one preferred embodiment, the fungal histidine kinase is a Neurospora crassa histidine kinase. In another preferred embodiment, the host cell expresses Candida albicans histidine kinase. In an alternative preferred embodiment, the host cell expresses Saccharomyces histidine kinase.
The present invention provides compositions and methods related to osmosensing fungal histidine kinases. In particular, the present invention provides amino acid and nucleic acid sequences of fungal histidine kinases from organisms such as Candida (e.g., C. albicans) and Neurospora (e.g., N. crassa). The present invention further provides compositions and methods for the development of antifungal compounds.
The present invention relates to fungal histidine kinases and methods for their use in screening compounds for antifungal activity, as well as for the development of antifungal compounds. Furthermore, the present invention provides compositions and methods for the treatment of fungal infections.
In one embodiment, the present invention provides a histidine kinase polypeptide from the fungal species Aspergillus fumigatus, named Fos-1p (SEQ ID NO: 35). The present invention also provides a second polypeptide encoded on the FOS-1 transcription unit, named mini-FOS-1 uORF (SEQ ID NO: 48). In related embodiments, the present invention provides compositions comprising the amino acid sequences selected from the group consisting of SEQ ID NOS: 35 and 48. In another related embodiment, the present invention provides nucleotide sequences encoding the polypeptides of SEQ ID NOS: 35 and 48. In another related embodiment, the present invention provides compositions comprising the nucleotide sequences encoding the polypeptides of SEQ ID NOS: 35 and 48.
In another embodiment, the present invention provides nucleotide sequences corresponding to genomic and cDNA nucleotide sequence from the A. fumigatus FOS-1 locus (SEQ ID NOS: 34 and 36, respectively), the Fos-1p open reading frame (SEQ ID NO: 59) and the mini FOS-1 uORF (SEQ ID NO: 47), and their complementary sequences. In a related embodiment, the present invention provides compositions comprising these genomic and cDNA nucleotide sequences. In another related embodiment, the present invention provides vectors comprising these nucleotide sequences. In an alternative embodiment, it is contemplated that these vectors are expression vectors. In a related embodiment, the vectors of the present invention are contained within a host cell. In one embodiment, the host cell is prokaryotic, while in another embodiment, the host cell is eukaryotic.
In another embodiment, the present invention provides a pure, isogenic strain of the fungal species Candida albicans having homozygous deletion of the endogenous loci encoding the amino acid sequence of Cos-1p (SEQ ID NO:17). Similarly, the present invention also provides a pure, isogenic strain of the fungal species Aspergillus fumigatus having homozygous deletion of the endogenous loci encoding the amino acid sequences of Fos-1p and mini-FOS-1 uORF polypeptide (SEQ ID NOS: 35 and 48, respectively).
The present invention provides antibodies directed against the C. albicans histidine kinase Cos-1p (SEQ ID NO: 17), the A. fumigatus histidine kinase Fos-1p (SEQ ID NO: 35), and the A. fumigatus mini-FOS-1 uORF polypeptide (SEQ ID NO: 48). The antibody of the present invention is selected from the group consisting of monoclonal antibodies and polyclonal antibodies.
The present invention also provides polynucleotide sequences that hybridize under stringent conditions to the nucleotide sequence of C. albicans COS-1 (SEQ ID NO: 16) and A. fumigatus FOS-1 cDNA (SEQ ID NO: 36).
In other embodiments, the present invention provides methods for identifying compounds which are candidates for development as antifungal drugs. In one embodiment, a method is provided to identify compounds which are antifungal drug candidates comprising the testing of a compound for the ability to inhibit in vitro histidine and/or aspartate kinase activities of a purified fungal histidine kinase protein. In an alternative embodiment of this method, the amino acid sequence of the fungal histidine kinase protein is selected from SEQ ID NOS: 2, 5, 17 and 35.
In an alternative embodiment, another method is provided to identify compounds which are antifungal drug candidates comprising the testing of a compound for the ability to inhibit in vitro histidine and/or aspartate kinase activities of a fungal histidine kinase protein and the ability of that same compound to inhibit or prevent fungal growth in culture. In another related embodiment, an alternative method is provided to identify compounds which are antifungal drug candidates comprising testing the ability of a compound to inhibit in vitro histidine and/or aspartate kinase activities of a fungal histidine kinase protein, testing the ability of that same compound to inhibit or prevent fungal growth in culture, and testing the ability of the compound to suppress or prevent in vivo candidosis/candidemia in a non-human animal. In a related method, the in vivo candidosis/candidemia is in a mouse.
In another embodiment, the method to identify compounds which are antifungal drug candidates uses a fungal strain that is selected from the Neurospora crassa, Candida albicans and Aspergillus fumigatus. In still another embodiment, the method to identify compounds which are antifungal drug candidates uses a fungal strain selected from a wild-type Neurospora crassa strain, a wild-type Candida albicans strain, a wild-type Aspergillus fumigatus strain, a pure, isogenic strain of Neurospora crassa having homozygous deletion of the endogenous loci encoding the amino acid sequence set forth in SEQ ID NO: 2, a pure, isogenic strain of Candida albicans having homozygous deletion of the endogenous loci encoding the amino acid sequence set forth in SEQ ID NO: 17, and a pure, isogenic strain of Aspergillus fumigatus having homozygous deletion of the endogenous loci encoding the amino acid sequences set forth in SEQ ID NOS: 35 and 48.
In related embodiments, the present invention also provides compounds which are antifungal drug candidates, wherein said compound (i) inhibits in vitro kinase activity of a histidine kinase protein; or (2) inhibits in vitro kinase activity of a histidine kinase protein and inhibits fungal growth in culture; or (3) inhibits in vitro kinase activity of a histidine kinase protein, inhibits fungal growth in culture and inhibits in vivo candidosis/candidemia in a mouse.
In another embodiment, the present invention provides synthetic polypeptides which encompass the phosphorylation sites of a fungal histidine kinase, where the phosphorylation site is a histidine phosphorylation site or an aspartate phosphorylation site. In a related embodiment, the synthetic peptides comprise the phosphorylation sites provided in SEQ ID NOS: 41 and 42.
In another embodiment, the present invention provides purified, synthetic oligonucleotides consisting of between 12 and 200 nucleotides having antisense complementarity to the nucleotide sequence of a fungal histidine kinase gene. In a related embodiment, the invention provides oligonucleotides having antisense complementarity to the nucleotide sequence selected from the group consisting of SEQ ID NOS: 16, 34 and 36.
In another embodiment, the present invention provides a method for treating a subject for the purpose of eradicating, mitigating or preventing a fungal infection. This method provides a subject, an antifungal agent selected from the group consisting of (i) a monoclonal or polyclonal antibody directed against a fungal histidine kinase protein, (ii) a compound which is an antifungal drug candidate selected from the group consisting of a compound which inhibits the in vitro kinase activity of a histidine kinase protein, a compound which inhibits the in vitro kinase activity of a histidine kinase protein and inhibits fungal growth in culture, and a compound which inhibits the in vitro kinase activity of a histidine kinase protein, inhibits fungal growth in culture, and inhibits in vivo candidosis/candidemia in a mouse, (iii) an isolated, synthetic peptide, wherein said peptide comprises the amino acid sequences of a histidine phosphorylation site or an aspartate phosphorylation site, and (iv) a synthetic antisense oligonucleotide having complementarity to the nucleotide sequence of a fungal histidine kinase, and finally, a means of delivery of said antifungal agent to the subject. In various embodiments of this method, the subject may be displaying pathology resulting from a fungal infection, may be suspected of displaying pathology resulting from a fungal infection, or be at risk of displaying pathology resulting from a fungal infection. In still other embodiments of this method, the delivery of the antifungal agent may be systemic, localized or topical.
In summary, the present invention provides compositions and methods related to fungal histidine kinases. In particular, the present invention provides amino acid and nucleotide sequences of fungal histidine kinases from organisms such as Candida (e.g., C. albicans), Neurospora (e.g., N. crassa), and Aspergillus (e.g., A. fumigatus). The present invention further provides compositions and methods for the identification and development of antifungal compounds, as well as the treatment of subjects to eradicate, mitigate or prevent fungal infections.